过表达EP4基因的人CD34+细胞构建及EP4A对其在小鼠体内的归巢影响OA

Objective To construct a lentiviral vector for over-expressing the prostaglandin E receptor 4(EP4)gene,clone the exogenous EP4 gene into human CD34+cells,observe the homing of these EP4 gene over-expressing cells in mice,and preliminarily investigate the effect of the EP4-specific agonist(EP4A)on this homing.Methods ①An EP4-cDNA fragment was chemically synthesized and cloned into a GL107 plasmid to construct the GL107-EP4+plasmid.②The GL107-EP4+plasmid and a control GL107 plasmid were packaged into lentiviruses using 293T cells.③Human CD34+cells were infected with these lentiviruses.Infection efficiency was assessed via fluorescence microscopy and flow cytometry,while EP4 expression was detected by qRT-PCR and Western blot.④The effect of EP4A on the homing of EP4+-human-CD34+cells in mice and the determination of the appropriate observation time was observed using in vivo imaging.⑤Detect the expression of EP4 in the bone marrow of mice after human CD34+cells were transfused by immunohistochemistry after EP4A stimulation.⑥CXCR4 expression in the bone marrow cells of these mice was measured by qRT-PCR and Western blot.Result ①DNA sequencing confirmed the correct EP4-cDNA sequence in the GL107-EP4+plasmid.②The GL107-EP4+plasmid was successfully packaged into lentiviruses with a titer＞1×108TU/mL.③Fluorescence microscopy observed that the GFP fluorescence intensity of GL107-EP4+and GL107 lentivirus-infected human CD34+cells was enhanced compared to the background control(uninfected lentivirus human CD34+cells);flow cytometry detected the infection efficiency of GL107-EP4+and GL107 lentivirus as 28.06%and 66.76%,respectively.The expression of EP4 messenger RNA(mRNA)and protein in GL107-EP4+was higher than in background control and GL107 lentivirus groups(0.580±0.032 vs.0.256±0.027 vs.0.250±0.043,both P＜0.001).④Day 3 post-transplantation was identified as the appropriate observation time for the EP4A-enhanced homing effect(P＜0.05).⑤The relative expression level of EP4 in the bone marrow of the EP4A-stimulated group after transplantation is higher than that of the EP4+-Luc control group(0.164±0.004 vs.0.118±0.007,P=0.016).⑥Both CXCR4 mRNA and protein levels were higher in the EP4A-stimulated group(both P＜0.05).Conclusion The EP4 gene was successfully cloned into human CD34+cells,leading to EP4 protein over-expression.EP4A may increase the directional homing efficiency of human CD34+hematopoietic cells to the bone marrow of recipient mice by promoting the expression of CXCR4.